SUMMARY Erythroid cells mature in vivo in the presence of a supportive macrophage; this cell-cell compartment is known as the erythroblastic island, an entity first described over 50 years ago. Red cell interactions with the macrophage are critical for optimal nutrient access, survival, proliferation, and differentiation of the erythron. Our proposed experiments address the exciting idea that Erythroid Krppel-like Factor (EKLF; KLF1) not only plays an intrinsic role in establishing the proper gene expression patterns in the red cell within the erythroblastic island, but that it additionally affects this process by an extrinsic mechanism based on its surprising expression within the island macrophage and early in development within the erythro-myeloid progenitor (EMP). Our studies build on observations made in primary or minimally manipulated cells, aided by in vivo assays and EKLF rescue systems. We have found that EKLF is expressed in the EMP in the yolk sac by E8.5. The experiments of Aim 1 will investigate the role of EKLF in directing the functional potential of these cells at later stages. We have found that macrophage-restricted ablation of EKLF affects the red cell, and that the EKLF-expressing fetal liver macrophage may express a unique expression signature. The experiments of Aim 2 will examine an iron-related hypothesis for the red cell effect, and will identify and assess EKLF's role in macrophage gene regulation. We found that EKLF plays a directive role in enucleation due to its control of cell cycle genes, especially cell division cycle inhibitors at late differentiation stages. The experiments of Aim 3 will evaluate how EKLF control of early or late expressing genes alters island formation, and will examine how cell cycle inhibitors exert their effect on enucleation in the absence of EKLF. Understanding these basic mechanisms will ultimately aid in understanding the problems that arise in anemias that present with high levels of nucleated red blood cells in circulation, such as congenital dyserythropoietic anemia (CDA) type IV, and in the design of culture systems that enable efficient expansion and enucleation of human cell sources for clinical use.